Martin Crompton

This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death. The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes. In vitro, under pseudopathological conditions of oxidative stress, relatively high Ca2+ and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-Mr solutes across the inner membrane. These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia/reperfusion. Evidence that the pore does open during ischaemia/reperfusion is discussed. There are also strong indications that the VDAC-adenine nucleotide translocase-CyP-D complex can recruit a number of other proteins, including Bax, and that the complex is utilized in some capacity during apoptosis. The apoptotic pathway is amplified by the release of apoptogenic proteins from the mitochondrial intermembrane space, including cytochrome c, apoptosis-inducing factor and some procaspases. Current evidence that the pore complex is involved in outer-membrane rupture and release of these proteins during programmed cell death is reviewed, along with indications that transient pore opening may provoke 'accidental' apoptosis.

This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death.The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes.In itro, under pseudopathological conditions of oxidative stress, relatively high Ca# + and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-M r solutes across the inner membrane.These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia\reperfusion.Evidence that the pore does open during ischaemia\reperfusion is discussed.There are also 1 Ca 2 + CYCLING AND THE CONTROL OF MITOCHONDRIAL Ca 2 + PT-induced mitochondrial dysfunction is a consequence of mitochondrial Ca# + overload.It is appropriate, therefore, to define the conditions under which the overload takes place.In the following, mitochondrial Ca# + overload is discussed against a background of how mitochondrial Ca# + is controlled normally, and how it can lead to PT pore opening.

A cyclophilin-D affinity matrix was employed to isolate components of the mitochondrial permeability transition pore. A cDNA encoding cyclophilin-D was cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S-transferase/cyclophilin-D fusion protein in Escherichia coli XL1 cells. The cyclophilin-D in the fusion was functionally normal as judged by its peptidylprolyl cis-trans-isomerase activity and its inhibition by cyclosporin A. The fusion protein was bound to glutathione-agarose to form the cyclophilin-D affinity matrix. The matrix selectively bound 32-kDa proteins of mitochondrial membrane extracts, but no H2O-soluble proteins were bound. The 32-kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage-dependent anion channel (porin) and the adenine nucleotide translocase. These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A. The thus-purified voltage-dependent anion channel, adenine nucleotide translocase and the fusion protein were incorporated into phosphatidylcholine liposomes containing fluorescein sulphonate. The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A. These properties are identical to those of the permeability transition pore in mitochondria. It is concluded that the basic permeability transition pore structure comprises the voltage-dependent anion channel (outer membrane), adenine nucleotide translocase (inner membrane) and cyclophilin-D, and forms at contact sites between the two membranes.

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits the further uptake of Ca 2+ by these mitochondria but induces little or no net Ca 2+ efflux; the further addition of Na + , however, induces rapid efflux of Ca 2+ . The velocity of the Na + ‐induced efflux of Ca 2+ from these mitochondria exhibits a sigmoidal dependence on the [Na + ]. Addition of Na + to mitochondria exhibiting the most active Na + ‐dependent efflux of Ca 2+ (brain and adrenal cortex) also releases Ca 2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca 2+ from these mitochondria occurs via a Na + ‐dependent pathway, possibly a Na + ‐Ca 2+ antiporter, that is distinct from the ruthenium‐red‐sensitive carrier that catalyses energy‐linked Ca 2+ influx. The possible role of the Na + ‐dependent efflux process in the distribution of Ca 2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na + ‐dependent efflux of Ca 2+ .